Characterizing the TCRα and TCRβ chains expressed by T cells responding to a given pathogen or underlying autoimmunity helps in the development of vaccines and immunotherapies, respectively. However, our understanding of complementary TCRα and TCRβ chain utilization is very limited for pathogen-and autoantigen-induced immunity. To address this problem, we have developed a multiplex nested RT-PCR method for the simultaneous amplification of transcripts encoding the TCRα and TCRβ chains from single cells. This multiplex method circumvented the lack of antibodies specific for variable regions of mouse TCRα chains and the need for prior knowledge of variable region usage in the TCRβ chain, resulting in a comprehensive, unbiased TCR repertoire analysis with paired coexpression of TCRα and TCRβ chains with single-cell resolution. Using CD8 + CTLs specific for an influenza epitope recovered directly from the pneumonic lungs of mice, this technique determined that 25% of such effectors expressed a dominant, nonproductively rearranged Tcra transcript. T cells with these out-of-frame Tcra mRNAs also expressed an alternate, in-frame Tcra, whereas approximately 10% of T cells had 2 productive Tcra transcripts. The proportion of cells with biallelic transcription increased over the course of a response, a finding that has implications for immune memory and autoimmunity. This technique may have broad applications in mouse models of human disease. IntroductionRecent advances have allowed us to analyze the development and persistence of virus-specific CD8 + T cell-mediated immunity from naive CTL precursors (CTLps) in peripheral lymphoid tissue, through the antigen-driven phase in lymph nodes and spleen, to the CTL effectors in a site of virus-induced pathology, and then, ultimately, to the persistence and recall of immune memory (1-3). However, unless we use lymphocytes from TCR-transgenic mice, our capacity to follow the fate and persistence of defined clonotypes is very limited. Several approaches have been used to estimate the extent of TCR diversity and to track clonally expanded T cell populations throughout the course of antigen-specific CTL responses (4), but none has given the complete picture. A commonly used protocol is to double-stain CD8 + T cells with mAbs specific for TCR variable (V) region β (TRBV) and tetramers specific for peptide + class I MHC glycoprotein (pMHCI) epitopes (5-9). Such low-resolution analysis provides no insight into the extent of clonal diversity within a particular TRBV-specific population and offers little scope for determining the spectrum of TCRα usage, as there are few mAb reagents. Another approach, known as immunoscope or spectratyping, uses gel electrophoresis of total mRNA from TRBV-specific populations (RT-PCR product) to determine profiles of complementarity-determining region 3β (CDR3β) length (4, 10). Combining spectratyping with cloning and sequencing allows for more definitive identification of CDR3β transcripts, but the approach is compromised by the possibility of bias durin...

Highly pathogenic avian influenza viruses pose a continuing global threat. Current vaccines will not protect against novel pandemic viruses. Creating “universal” vaccines has been unsuccessful because the immunological mechanisms promoting heterosubtypic immunity are incompletely defined. We show that rapamycin, an immunosuppressive drug that inhibits mTOR, promotes cross-strain protection against lethal H5N1 and H7N9 infections when administered during H3N2 virus immunization. Rapamycin reduced germinal center formation and inhibited B cell class-switching, yielding a unique repertoire of antibodies that mediated heterosubtypic protection. Our data establish a requirement for mTORC1 in B cell class-switching and demonstrate that rapamycin skews the antibody response away from high affinity variant epitopes, targeting more conserved elements of hemagglutinin. These findings have intriguing implications for influenza vaccine design.

It is currently thought that T cells with specificity for self-peptide/ MHC (pMHC) ligands are deleted during thymic development, thereby preventing autoimmunity. In the case of CD4 + T cells, what is unclear is the extent to which self-peptide/MHC class II (pMHCII)-specific T cells are deleted or become Foxp3 + regulatory T cells. We addressed this issue by characterizing a natural polyclonal pMHCII-specific CD4 + T-cell population in mice that either lacked or expressed the relevant antigen in a ubiquitous pattern. Mice expressing the antigen contained one-third the number of pMHCII-specific T cells as mice lacking the antigen, and the remaining cells exhibited low TCR avidity. In mice lacking the antigen, the pMHCII-specific T-cell population was dominated by phenotypically naive Foxp3− cells, but also contained a subset of Foxp3 + regulatory cells. Both Foxp3 − and Foxp3 + pMHCII-specific T-cell numbers were reduced in mice expressing the antigen, but the Foxp3 + subset was more resistant to changes in number and TCR repertoire. Therefore, thymic selection of self-pMHCII-specific CD4 + T cells results in incomplete deletion within the normal polyclonal repertoire, especially among regulatory T cells.ccording to clonal selection theory, self-reactive lymphocytes are deleted during development, thereby ensuring immunological tolerance to self tissues (1). In the case of T cells, this process occurs in the thymus where developing thymocytes with randomly generated T-cell antigen receptor (TCR) specificities are educated on an array of self-peptide/MHC (pMHC) ligands (2). In the simplest form of this model, thymocytes receiving strong TCR signals are deleted, thereby purging the repertoire of any cells with self-pMHC specificity. The significance of this mechanism of central tolerance has been substantiated by studies demonstrating that impairment of antigen presentation in the thymus can lead to autoimmunity (3, 4). Nevertheless, the clonal deletion rule is challenged by the wellknown observation that immunization with certain self-peptides can induce autoimmunity, indicating that at least some responsive self-pMHC-specific T cells routinely exist in the repertoire (5).Another intriguing exception to the clonal deletion rule is presented by regulatory T (Treg) cells, a suppressive subset of CD4 + T cells defined by expression of the Foxp3 transcription factor (6, 7). Whereas some Treg cells are induced from conventional T cells during immune responses (iTreg), most are socalled natural regulatory T (nTreg) cells, which arise directly in the thymus much like naive CD4 + T cells (8). Studies have shown that nTreg cells develop as a consequence of the type of high-affinity TCR-self-pMHCII interaction that was thought to cause clonal deletion (9). Moreover, extensive TCR sequencing studies of Treg and non-Treg populations have indicated that, whereas the specificities of these repertoires overlap, Treg cell specificity is biased toward self-pMHCII ligands (10, 11).These findings lead to the question of why some sel...

The A/VN/1203/04 H5N1 influenza virus is capable of infecting the CNS of mice and inducing a number of neurodegenerative pathologies. Here, we examined the effects of H5N1 on several pathological aspects affected in parkinsonism, including loss of the phenotype of dopaminergic (DAergic) neurons located in the substantia nigra pars compacta (SNpc), expression of mono- and indolamines in brain, alterations in SNpc microglia number and morphology, and expression of cytokines, chemokines and growth factors. We find that H5N1 induces a transient loss of the DAergic phenotype in SNpc and now report that this loss recovers by 90 days post infection (dpi). A similar pattern of loss and recovery was seen in monoamine levels of the basal ganglia. The inflammatory response in lung and different regions of the brain known to be targets of the H5N1 virus (brainstem, substantia nigra, striatum, and cortex) were examined at 3, 10, 21, 60 and 90 dpi. We found a significant increase in the number of activated microglia in each of these brain regions that lasted at least 90 days. We also quantified expression of IL-1α, IL-1β, IL-2, IL-6, IL-9, IL-10, IL-12(p70), IL-13, TNF-α, IFN-γ, GM-CSF, G-CSF, M-CSF, eotaxin, IP-10, KC, MCP-1, MIP-1α, MIP-1β and VEGF and find that the pattern and levels of expression are dependent on both brain region and time after infection. We conclude that H5N1 infection in mice induces a long-lasting inflammatory response in brain and may play a contributing factor in the development of pathologies in neurodegenerative disorders.

Following respiratory syncytial virus infection of adult CB6F1 hybrid mice, a predictable CD8+ T cell epitope hierarchy is established with a strongly dominant response to a Kd-restricted peptide (SYIGSINNI) from the M2 protein. The response to KdM282-90 is ∼5-fold higher than the response to a subdominant epitope from the M protein (NAITNAKII, DbM187-195). After infection of neonatal mice, a distinctly different epitope hierarchy emerges with codominant responses to KdM282-90 and DbM187-195. Adoptive transfer of naïve CD8+ T cells from adults into congenic neonates prior to infection indicates that intrinsic CD8+ T cell factors contribute to age-related differences in hierarchy. Epitope-specific precursor frequency differs between adults and neonates and influences, but does not predict the hierarchy following infection. Additionally, dominance of KdM282-90 –specific cells does not correlate with TdT activity. Epitope-specific Vβ repertoire usage is more restricted and functional avidity is lower in neonatal mice. The neonatal pattern of codominance changes after infection at 10 days of age, and rapidly shifts to the adult pattern of extreme KdM282- 90 -dominance. Thus, the functional properties of T cells are selectively modified by developmental factors in an epitope-specific and age-dependent manner.

One question that continues to challenge influenza A research is why some strains of virus are so devastating compared to their more mild counterparts. We approached this question from an immunological perspective, investigating the CD8 ؉ T cell response in a mouse model system comparing high-and low-pathological influenza virus infections. Our findings reveal that the early (day 0 to 5) viral titer was not the determining factor in the outcome of disease. Instead, increased numbers of antigen-specific CD8؉ T cells and elevated effector function on a per-cell basis were found in the low-pathological infection and correlated with reduced illness and latertime-point (day 6 to 10) viral titer. High-pathological infection was associated with increased PD-1 expression on influenza virus-specific CD8؉ T cells, and blockade of PD-L1 in vivo led to reduced virus titers and increased CD8 ؉ T cell numbers in high-but not lowpathological infection, though T cell functionality was not restored. These data show that high-pathological acute influenza virus infection is associated with a dysregulated CD8 ؉ T cell response, which is likely caused by the more highly inflamed airway microenvironment during the early days of infection. Therapeutic approaches specifically aimed at modulating innate airway inflammation may therefore promote efficient CD8 ؉ T cell activity. IMPORTANCEWe show that during a severe influenza virus infection, one type of immune cell, the CD8 T cell, is less abundant and less functional than in a more mild infection. This dysregulated T cell phenotype correlates with a lower rate of virus clearance in the severe infection and is partially regulated by the expression of a suppressive coreceptor called PD-1. Treatment with an antibody that blocks PD-1 improves T cell functionality and increases virus clearance.

While differences in the rate of virus fusion and budding from the host cell membrane have been correlated with pathogenicity, no systematic study of the contribution of differences in viral envelope composition has previously been attempted. Using rigorous virus purification, marked differences between virions and host were observed. Over 125 phospholipid species have been quantitated for three strains of influenza (HKx31- H3N2, PR8- H1N1, and VN1203- H5N1) grown in eggs. The glycerophospholipid composition of purified virions differs from that of the host or that of typical mammalian cells. Phosphatidylcholine is the major component in most mammalian cell membranes, while in purified virions phosphatidylethanolamine dominates. Due to its effects on membrane curvature, it is likely that the variations in its content are important to viral processing during infection. This integrated method of virion isolation with systematic analysis of glycerophospholipids provides a tool for the assessment of species specific biomarkers of viral pathogenicity.

During influenza virus infection, it is unclear how much alveolar cell loss can be tolerated before the host succumbs to the disease. We sought to define relevant correlates of disease severity in the mouse influenza model, hypothesizing that a susceptibility threshold exists for alveolar epithelial cell loss. We compared lung pathology, virus spread, alveolar epithelial cell depletion, arterial blood oxygenation, physiological responses measured by unrestrained plethysmography, and oxygen consumption and carbon dioxide production by gas analysis in mice at intervals after infection with virus strains and doses that cause mild (x31) or severe (PR/8) influenza. Both mild and severe infections showed similar degrees of lung damage and virus dissemination until day 6 after inoculation but diverged in survival outcomes from day 9. Day 6 PR/8-infected mice had normal respiratory and gas exchange functions with 10% type I cell loss. However, day 10 PR/8-infected mice had 40% type I cell loss with a concomitant drastic decreases in tidal and minute volumes, Vo(2), Vco(2), and arterial blood oxygenation, compared with a maximum 3% type I cell loss for x31 on day 10 when they recovered body weight and respiratory functions. Alterations in breaths per minute, expiratory time, and metabolic rate were observed in both infections. A threshold for maintenance of proper respiratory function appears to be crossed once 10% of alveolar type I cells are lost. These data indicate that lethality in influenza virus infection is a matter of degree rather than quality.